In successful models of transplantation tolerance, harmful donor-specific responses in the recipient are durably eliminated and the allograft is maintained for the life of the recipient. In this project, we propose to study the manipulation of donor stem cells on host immune responses as, given the proper conditions. These cells have the qualities of self-renewal, and could potentially provide lifetime immunoregulation. In the first specific aim, we will test whether donor hematopoietic stem cell grafts can be engineered to exert a tolerogenic effect on host immune responses by manipulating stem cell and non-stem cell subpopulations. This proposal is based on our observations in which cynomolgus monkeys recipients of MHC-mismatched mobilized peripheral blood stem cell grafts had longer periods of chimerism (399 +/- 88 vs.103 +/- 37 days) and renal allograft survival (more than 145 vs. 18 +/- 15 days) when compared to bone marrow stem cell recipients with the same content of CD34+ cells, suggesting more effective immunoregulation exerted by the sub-populations within the mobilized peripheral stem cell graft. We will also test whether donor specific hyporesponsiveness via mixed lymphocyte reaction, cytokine profiling, and the absence of anti-donor antibody correlates with the acceptance of renal allografts. The second specific aim will test whether the stem cells of the bone marrow microenvironment can be manipulated to further enhance the graft survival observed following mobilized stem cell administration. Mesenchymal stem cells can be induced to differentiate into the components of the bone marrow stromal microenvironment, such as bone. These stromal elements can provide regulatory signals that inhibit or promote Iympho- and myelopoiesis, differentiation, proliferation, and T and B cell migration, and thus, have the potential to affect the transplanted organ. Our studies have revealed that mesenchymal stem cells can prolong skin graft survival in baboons and intact active bone grafts can permit permanent acceptance of cardiac allografts in mice. We will test which component of the microenvironment provides the greatest immunomodulatory effect. The degree of recipient conditioning may also playa role in the induction of tolerance. The third approach will be focused on residual host mature T and B cells thought to have escaped the non-radiation based, non-myeloablative conditioning regimen used in our cynomolgus monkey model of stem cell transplantation. We have observed some recipients to develop anti-donor antibody detected briefly between the 2nd and 4th months and hypothesize that the emergence of this antibody indicates residual alloreactivity that will ultimately impede the maintenance of tolerance. We will test whether residual alloreactivity can be eliminated by T cell inactivation, through a brief course of post-transplant cyclosporine, or through B cell depletion using an anti-CD20 monoclonal antibody. We anticipate the combined insights obtained through the manipulation of stem cell contents in specific aims I and 2 and mature host responses specific aim 3, will lead to the development a novel regimen for the induction of tolerance to renal allografts.